We demonstrate how photoinduced electron transfer (PET) reactions can be successfully applied to monitor conformational dynamics in individual biopolymers. We present data about structural changes of single biomolecules like peptides by using quenching electron transfer reactions between tryptophan residues in close proximity to fluorescent dyes. Our results demonstrate that selective PET-reactions between fluorophores and amino acids represent a versatile tool to measure small-scale conformational dynamics in biopolymers on a wide range of time scales, extending from nanoseconds to seconds, at the single-molecule level under equilibrium conditions. That is, the monitoring of conformational dynamics of biopolymers with temporal resolutions comparable to those within reach using new techniques of molecular dynamic simulations. Furthermore, we demonstrate that the strong distance dependence of charge separation reactions on the sub-nanometer scale can be used to develop conformationally flexible PET-biosensors. These sensors enable the detection of specific target molecules in the sub-picomolar range and allow one to follow their molecular binding dynamics with temporal resolution.

The optical microscope has proven to be an invaluable tool in biology, because of its unique capabilities of 3-D imaging of living specimens. However, compared to other techniques, the achievable resolution is limited. Several techniques have been proposed to improve the resolution of the fluorescence microscope. The confocal set-up is the first of them. Interference effects can also be used to sharpen up the point spread function (PSF) in 4Pi microscopy. Another approach is the so-called STimulated Emission Depletion microscopy, which has permitted to decrease the resolution down to about 100 nm in three dimensions, and below 50 nm in either the x-y plane, or along the z-axis.

Optical elevators are constructed from counter-propagating coaxial beams with orthogonal linear polarizations, the strengths of which are controlled by a spatial polarization modulator. Each of these optical potential wells draws a dielectric particle towards the optical axis where it finds a stable transverse position. Adjustment of the relative strengths of the counteracting scattering forces induced by the opposing beams enables control over the axial trap position of the particle within the confines of the optical elevator. We have devised a power-preserving scheme using a spatial polarization modulator to vary the intensity ratio of the two polarization states. The proposed method has the potential of extending the dynamic range in axial positioning of optically trapped particles surpassing the range offered by other techniques.

Sequence specific cutting of DNA is a standard method in molecular biology. This cutting is realized with enzymes which have a defined recognition sequence and cutting sequence. Therefore one can manipulate only sequences for which an enzyme is available. With current physical methods (AFM) any sequences can be cut, but the precise sequence specific and highly parallel cutting is not possible. Near infrared (NIR) femtosecond laser systems have been used to optically knock out genomic regions of highly condensed DNA in human chromosomes as well as of single expanded (stretched) DNA molecules. Working with 80 MHz laser pulses at 800 nm of low 2 nJ pulse energy but at high TW/cm² light intensities, multiphoton ionization and optical breakdown (OB) resulted in highly precise material ablation with sub-100 nm cut sizes. This is far below the diffraction-limited spot size. A minimum FWHM cut size of 65 nm was achieved in the case of the nanodissection of a laser-treated stretched λ-DNA (48kb) molecule which corresponded to 200 optically knocked out bases. By the use of metal nanoparticles as energy coupling objects for fs laser radiation we expect a specific highly local destruction effect within the DNA molecule (cut). Thereby, a sequence-specific binding of DNA nanoparticle complexes along the target DNA is a fundamental condition. The effect of laser exposure on DNA and DNA-nanoparticle complexes are presented.

Recently medical visualization systems require extensive support from stereo imaging technologies in order to increase effectiveness of recognitions of low contrast micro biological structures. Low image contrast prevents identifications of tissues and in many cases is a reason of forbear from operation. In this paper authors present a system consisting of stereoscopic microscope with two image acquisition channels, data processing unit and Helmet Mounted Display (HMD) for stereoscopic visualization of operational field. Special attention is paid to the development of automatic image segmentation and low contrast areas enhancement algorithms. Algorithms proposed automatically find regions characterized by low contrast, modify intensity distribution and display enhanced stereo images in real time. The conditions of images processing and manipulation in order to assure synchronization of images transfer in both visual channels are presented. The algorithms are optimized for stereo visualization of biological micro-structures of inner ear. Some of those structures have quasi-phase nature and are almost invisible by human eye. The tests performed at the images captured during operation have been positively evaluated by surgeons. The performance of the system presented ascertained that quality of inner ear processed images guaranties safety carrying of operations not undertaken so far.

A microscopic birefringence imaging of bio-sample is proposed. This system consists of a super luminescent diode (SLD), polarizers, a quarter-wave plate and a phase retarder. The instrument is provided to map and visualize an optical anisotropy in bio-sample. A local-sampling phase shifting technique is employed for analytic algorithm with high resolution of retardance. A Bereck compensator is used a sample for checking its accuracy. Birefringence distributions of gelatin orientation such as retardance and azimuthal direction are shown in case of applied voltage and changing temperature as its demonstration. It is possible to observe molecular orientation of bio-sample.

We propose a solution for increasing the axial resolution of confocal microscopes. In the experimental set-up described in this paper an interference phenomenon between two counterpropagating beams is used to determine the axial position of a luminophore. The optical path difference between the two waves, which is related to the position of the luminophore, is recovered thanks to a second interferometer by using partial coherence interferometry demodulation technique. The proposed solution can find applications in biology for localizing with nanometric resolution a small number of tagged species.

We have been developing a polarized light microscope with liquid crystal universal compensator and circular polarizer (the LC-PolScope) for recording images, which are independent of the orientation of birefringent objects. Separate images show the retardance and the slow axis azimuth distributions of the in-plane birefringence of the focused region in the specimen. However the measured (apparent) retardance still depends on the angle between the crystal optic axis and the axis of the illuminating beam of light. If the illuminating beam is close to parallel to the optic axis the measured retardance value decreases dramatically and becomes zero when the two axes are parallel. The description of birefringent objects oriented in 3-dimensional space requires the introduction of two additional parameters: the principal retardance and the inclination angle. Together with the azimuth angle they completely characterize the birefringence properties of a specimen, assuming the specimen has a uniaxial optical indicatrix. We devised a new technique for measuring the three birefringence parameters without moving the specimen. For exploring the out-of-plane birefringence the new instrument which is based on the LC-PolScope technique contains an additional spatial light modulator, implemented here as a liquid crystal mask. The mask is located in the aperture plane of the condenser lens. Partial occlusion of the condenser aperture changes the direction of the central ray of the cone of light converging on the specimen. So we can obtain the retardance and azimuth images using different sets of illumination rays. For experimental verification we used a biological object called an aster. An aster consists of nearly parallel arrays of microtubules, a stiff biopolymer, radiating from a common organizing center called a centrosome. The object is spherically symmetric, and its 3 dimensional distribution of birefringence orientation can be predicted. Experimental results have shown the developed polarizing microscope can successfully be used for imaging and measuring three-dimensional orientation of birefringent objects

In biological micromanipulation image aberrations are introduced not only by the optical system, but also by the immersion liquid. Whereas optical system aberrations are constant and it is relatively easy to measure and correct for them, the immersion caused aberrations are variable in time and space. In this paper a method using a spherical microparticle as an artificial point source for aberration control is presented. The particle is positioned by optical tweezers at the location of the biological sample. In the experiment holographic tweezers are used. They are based on computer generated holograms, written into spatial light modulators, which create light traps for the microparticle in the object plane. The light traps can be moved without any mechanically moving parts, just by changing the hologram. The particle strongly focuses the light, therefore an artificial point source in the object space is created. The illumination light is filtered, so that only the signal corresponding to a spherical wave is analyzed by the wavefront detection system. The information about the wavefront distortion is used to dynamically correct for it. This can be done by using spatial light modulators. The method is suitable for biophotonic imaging systems, where refractive index variations in the sample plane are significant. The integration with holographic tweezers is advantageous since it offers flexibility in positioning and imaging the particles.

In this study properties of diffuse light from a spherical media embedded in an infinite media was investigated. Light that migrates through the spherical media is considered as signal and light that does not propagate through the spherical media is considered as noise. The analytical solution of SNR (signal to noise ratio) was derived with diffusion theory. The spatial distributions of the fluence rate were analyzed and the contours of signal to noise ratio were obtained as light source was put in different positions. The relationship between the source detector separation corresponding to maximum SNR and light source position was discussed, which is helpful to determine an appropriate measurement position. The results acquired in this paper are useful for ultrasound-modulated optical tomography and tissue imaging with diffuse photon density waves.

We report highly efficient Forster resonance energy transfer between CdTe nanocrystals and two different dyes, Rhodamine B and Oxazine, where the nanocrystals are mixed with the dyes on top of glass substrates. A faster NC decay curve is observed in the samples containing NCs mixed with dyes than in those containing NCs on their own. For the samples containing nanocrystals mixed with Rhodamine B, room temperature PL measurements are presented as a function of the ratio between the amount of acceptors and the amount of donors, C_A/C_D. This ratio is varied between 0.03 and 5. The strongest enhancement of the acceptor PL intensity relative to that of the donor PL intensity is reached for 0.2A/C_D<5, suggesting that most efficient FRET is also achieved in this region.

Fluorescence correlation spectroscopy (FCS) is a mature and powerful technique for measuring diffusion coefficients. In a standard experiment, it measures the spontaneous fluorescence fluctuations arising from a single observation volume defined by confocal optics. However, the study becomes uneasy as soon as the diffusion is impeded by obstacles or specific mechanisms, as it is the case for the cell membrane components in live cells. In this paper, we show that doing FCS measurements at different sizes of observation volumes gives access to the diffusion laws without a priori knowledge of the landscape in which molecules are diffusing. Using this strategy, a measurement of diffusion laws of lipids in monophasic Giant Unilamellar Vesicles and in the plasma membrane of live cells is carried out.

Near infrared (NIR) ultrashort laser pulses of 780 nm have been used to induce intracellular photodynamic reactions by nonlinear excitation of porphyrin photosensitizers. Intracellular accumulation and photobleaching of the fluorescent photosensitizers protoporphyrin IX and Photofrin (PF) have been studied by non-resonant two-photon fluorescence excitation of PF and aminolevulinic acid (ALA)-labeled Chinese hamster ovary (CHO) cells. To testify the efficacy of both substrates to induce irreversible destructive effects, the cloning efficiency (CE) of cells exposed to femtosecond pulses of a multiphoton laser scanning microscope (40x/1.3) was determined. In the case of Photofrin accumulation, CEs of 50% and 0% were obtained after 17 laserscans (2 mW?, 16 s/ frame) and 50 scans, respectively. All cells exposed to 50 scans died within 48h after laser exposure. 100 scans were required to induce lethal effects in ALA labeled cells. Sensitizer-free control cells could be scanned 250 times (1.1 h) and more without impact on the reproduction behavior, morphology, and vitality. In addition to the slow phototoxic effect by photooxidation processes, another destructive but immediate effect based on optical breakdown was induced when employing high intense NIR femtosecond laser beams. This was used to optically knock out single tumor cells in living mice (solid Ehrlich-Carcinoma) in a depth of 10 to 100 μm.

A time-correlated single photon counting (TCSPC) module (SPC-730, Becker & Hickl, Germany) was connected to a laser scanning microscope (Zeiss, Germany) equipped with an ultrafast photomultiplier. Short pulse excitation was achieved with two laser diodes emitting at 398nm and 434nm with a pulse duration of 70ps and 60 ps (PicoQuant, Germany) to allow intracellular fluorescence lifetime imaging (FLIM). With this setup, fluorescence lifetime of the mitochondrial marker Rhodamine 123 could be studied in solution under the same instrumental conditions as used for fluorescence lifetime imaging of cell monolayers. With the same set of parameters, fluorescence lifetime of Rhodamine 123 was calculated with good reproducibility in mitochondria of living cells. We present here a comparison of different fitting routines, including a multiexponential fitting based on the method of Laplace transformation. Fluorescence lifetimes calculated with the multiexponential fitting routine proved to be particularly useful to study the distribution of 5-ALA metabolites in cell monolayers.

Development of fluorescence microscopic methods is limited by the application of new dyes, the response of which could be sensitive to different functional states in the living cells, and, in particular, to electrostatic potentials on their plasma membranes. Recently, we showed that newly designed 3-hydroxyflavone fluorescence dyes are highly electrochromic and show a strong two-band ratiometric response to electric dipole potential in lipid membranes. In the present report we extend these observations and describe a new generation of these dyes as electrochromic probes in biomembrane research. Modification of the membrane dipole potential was achieved by addition of 6-ketocholestanol (6-KC), cholesterol and phloretin. The dipole potential was also estimated by the reference probe di-8-ANEPPS. As an example, we show that on addition of 6-KC there occurs a dramatic change of the intensity ratio of the two emission bands, which is easily detected as a change of color. We describe in detail the applications of one of these dyes, PPZ8, to the studies of cells in suspension or attached to the glass surface. Confocal microscopy demonstrates strong preference of the probe for the cell plasma membrane, which allows us to apply this dye for studying electrostatic and other biomembrane properties. We demonstrate that the two-color response provides a direct and convenient way to measure the dipole potential in the plasma membrane. Applying PPZ8 in confocal microcopy and two-photon microspectroscopy allowed us to provide two-color imaging of the membrane dipole potential on the level of a single cell.